Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine (Sq) dye (λabs= 670 nm, λem= 700 nm). The dye self-assembles to form nonfluorescent nanoparticles (Dh = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the Sq dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the Sq nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.
This study demonstrates that pH-responsive polymers have a very high buffering capacity in their immediate vicinity, a phenomenon termed "nanobuffering". This can be exploited to dissociate local nanoscale pH from bulk solution pH. Herein, a series of pH-responsive polymers were conjugated to Protein-A to rationally manipulate the latter's binding affinity toward antibodies via nanobuffering (i.e., this interaction is pH dependent), independently of bulk solution pH. Moreover, the nanobuffering effect could be terminated using low concentrations of strong ionpairing salts, to achieve quantitative release of the antibodies from the bioconjugate. These complementary discoveries are showcased in the context of the development of a homogeneous affinity precipitation agent (i.e., a scavenger) for the purification of polyclonal immunoglobulin G and two monoclonal antibodies from cell culture supernatant. Indeed, while bulk solution pH was used to induce precipitation of the scavenger, maintaining local nanoscale pH via nanobuffering maximized binding interaction with the antibodies. A 2:1 binding stoichiometry was observed, which was similar to that observed for native protein. The scavenger could be recycled multiple times, and the purification protocol circumvented lengthy/tedious physical purification processes typically associated with mAb manufacturing. Overall, this study provides perspectives on the local nanoscale pH near pH-responsive polymers and establishes lines of thought for predictably manipulating or even terminating nanobuffering, to control the activity of proteins.
Direct monitoring of apoptotic progression is a major step forward for the early assessment of therapeutic efficacy of certain treatments and the accurate evaluation of the spread of a disease. Here, the regulatory role of glutathione (GSH) is explored as a potential biomarker for tracking apoptosis. For this purpose, a near- infrared (NIR) squaraine dye is introduced that is capable of sensing GSH in a ratiometric manner by switching its emission from NIR (690 nm) to visible region (560 nm). The favorable biocompatible attributes of the probe facilitated the real-time monitoring of apoptotic process in line with the conventional apoptotic assay. Furthermore, the robust nature of the probe was utilized for the quantitative estimation of GSH during different stages of apoptosis. Through this study, an easy and reliable method of assaying apoptosis is demonstrated, which can provide valuable insights in translational clinical research.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.